Recent studies have demonstrated that plasmid DNA can be used as a non-viral vector to deliver therapeutic genes to human cells and tissues as a way of curing and preventing diseases such as cystic fibrosis and AIDS. One of the technological challenges associated with gene therapy and DNA vaccination is the development of processes for the large-scale purification of plasmid DNA, capable of delivering a product that is safe, efficient and economic according to the requirements of regulatory agencies. Although plasmid DNA is produced by growing bacterial cells like the majority of recombinant proteins, the information relative to purification methods for their large-scale production is still scarce. Additionally, the existent laboratory-scale protocols are not adequate for the large-scale production of pharmaceutical grade plasmid DNA, due to technological, economic and safety reasons, since they use organic, mutagenic and toxic compounds (phenol, chloroform, ethidium bromide, cesium chloride) and animal derived enzymes (lysozyme, protease K and RNase). The use of these reagents in a process for the production of a product that is intended for human use always requires its complete removal from the end product. This removal then has to be demonstrated unequivocally by the manufacturer, a process that is often difficult to carry out. The use of some reagents may even be barred in the first place. Some plasmid purification processes have been developed on the basis of these laboratory protocols with the intention of producing a pharmaceutical-grade product at large-scale.
The challenge posed to the purification process is the removal of impurities such as lipopolysaccharides, RNA, denatured plasmid DNA and genomic DNA (gDNA), which have properties similar to those of plasmid DNA and which behave identically in most of the purification operations. The production and purification of plasmid DNA comprehends the usual steps of fermentation, primary isolation and purification. At laboratory scale, primary isolation is usually performed by the alkaline lysis method, developed to disrupt cells and denature genomic DNA and proteins that precipitate together with cellular debris and other impurities. At this stage the use of animal-derived enzymes such as lysozyme and RNase is common. Due to its animal origin, these components may introduce viral (or other) contamination in the product, and thus its use is not advisable.
The lysate obtained may be concentrated and partially purified using extraction with organic solvents (mixtures of phenol, chloroform and iso-amylic alcohol) and/or precipitation with adequate agents such as isopropanol, ethanol, poly(ethylene glycol) and ammonium acetate. One or two chromatography operations usually follow. In most cases anion exchange and gel filtration chromatography have been used; affinity and reversed phase chromatography applications have also been described. On the other hand, hydrophobic interaction chromatography, which is a powerful technique often used in the purification of therapeutic proteins, has not been used for the purification of plasmid DNA.